Exendin-4 is a 39 amino acid peptide which is produced by the salivary glands of the Gila monster (Heloderma suspectum) (Eng, J. et al., J. Biol. Chem., 267:7402-05, 1992). Exendin-4 is an activator of the glucagon-like peptide-1 (GLP-1) receptor, whereas it does not activate significantly the glucagon receptor.
The amino acid sequence of exendin-4 is shown as SEQ ID NO: 1
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2
Glucagon is a 29-amino acid peptide which is released into the bloodstream when circulating glucose is low. Glucagon's amino acid sequence is shown as SEQ ID NO 2.
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH
During hypoglycemia, when blood glucose levels drop below normal, glucagon signals the liver to break down glycogen and release glucose, causing an increase of blood glucose levels to reach a normal level. Hypoglycemia is a common side effect in diabetics who are treated with insulin due to elevated blood glucose levels. Thus, glucagon's most predominant role in glucose regulation is to counteract insulin action and maintain blood glucose levels.
Glucagon has an isoelectric point of approximately 7 and is therefore only poorly soluble (<0.2 mg/ml) in the pH range of 4-8. It is well soluble (>10 mg/ml) at pH values below 3 or above 9 (Bromer, W. W., Handbook of Experimental Pharmacology, Vol 66/1, 1983). Consequently, the currently available commercial solutions of glucagon (GlucaGen® HypoKit, Glucagon emergency rescue kit) are acidic and need to be prepared freshly before use due to the chemical and biophysical instability of glucagon in solution at low pH (Joshi, A. B. et al, Int. J. Ph. Sci., 203, 115-125, 2000).
The preparation of glucagon formulations with enhanced stability compared to the commercial kit solutions are described in patent applications WO9947160, WO12059762, US2011/0097386, US2011/0237510, US2011/049713, WO12012460, WO12122535, US2012/0071817, and WO13101749, the contents of which are herein incorporated by reference.
The preparation of stabilized analogues of glucagon is described in patent applications WO14016300, WO11049713, WO07056362, WO08086086, and WO09155257, the contents of which are herein incorporated by reference.
The use of 4-Thiazolylalanine in position 1 of a synthetic peptide has been described in WO07140284 for GLP-1 receptor agonists. Conversely, 4-Thiazolylalanine in the present invention surprisingly provides highly active glucagon receptor agonists with reduced activity at the GLP-1 receptor when compared to peptides that carry the natural histidine at position 1 (native glucagon).